Primorigen Biosciences (R) Abstract Primorigen Biosciences will use SBIR funds to develop a high-throughput toxicology assay system utilizing human ventricular cardiomyocytes (vCMs) differentiated from human induced pluripotent stem cells (hiPSCs). Phase I studies will convert the small-molecule based, cytokine-free CardioTotal differentiation technology into a 96-well based high-throughput format. Automated flow cytometry will be used to monitor mesoderm specification by staining for brachyury, and to monitor differentiation efficiency and yield by staining for cardiac Troponin T and cardiac Troponin I. Cell viability and yield will be determined by cytoxicity assay, and contractility measurements will be performed by video microscopy and automated image analysis. Using these assays, a library of 20 toxicants selected from the Tox21 library will be screened, first for effects upon differentiation following early exposure, and second for toxicity and contractility effects by late exposure of differentiated cardiomyocytes. In Phase II, the system will be scaled up for 384-well format, and screening will be applied to a larger portion of the Tox21 library. Additional pluripotent cell lines will be tested to establish robustness, and secondary screenings will be initiated to identify mechanisms of action. In Phase III, the system will be commercialized as an optimized, high-throughput cardiotoxicity screening system, provided as a service by Primorigen or customized for end-user internalization (for example at NIEHS NTP or EPA's ToxCast program).